Strategies for selecting Recombinant CHO cell lines for cGMP manufacturing: Realizing the potential in bioreactors

Manufacture of recombinant proteins from mammalian cell lines requires the use of bioreactor systems at scales of up to 20,000 L. The cost and complexity of such systems can prohibit their extensive use during the process to construct and select the manufacturing cell line. It is therefore common practice to develop a model of the production process in a small scale vessel, such as a shake‐flask, where lower costs, ease of handling, and higher throughput are possible. This model can then be used to select a small number of cell lines for further evaluation in bioreactor culture. Here, we extend our previous work investigating cell line construction strategies to assess how well the behavior of cell lines in such a shake‐flask assessment predicts behavior in the associated bioreactor production process. A panel of 29 GS‐CHO cell lines, all producing the same antibody, were selected to include a mixture of high and low producers from a pool of 175 transfectants. Assessment of this panel in 10 L bioreactor culture revealed wide variation in parameters including growth, productivity, and metabolite utilization. In general, those cell lines which were high producing in the bioreactor cultures had also been higher producing in an earlier shake‐flask assessment. However, some changes in rank position of the evaluated cell lines were seen between the two systems. A potential explanation of these observations is discussed and approaches to improve the predictability of assessments used for cell line selection are considered. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

The role of the cell cycle in determining gene expression and productivity in CHO cells

Understanding the relationships between cell cycle and protein expression is critical to the optimisation of media and environmental conditions for successful commercial operation of animal cell culture processes. Using flow cytometry for the analysis of the early phases of synchronised batch cultures, the dependency of product expression on cell cycle related events has been evaluated in a recombinant CHO cell line. Although the production of recombinant protein is initially found to be cell cycle related, the maximum specific protein productivity is only achieved at a later stage of the exponential phase which also sees a maximum in the intracellular protein concentration. Subsequent work suggests that it is the batch phase/medium composition of cultures which is the major determinant of maximum specific productivity in this cell line. Furthermore the effect of the positive association between S phase and specific productivity is subordinate to the effect of batch phase/medium composition on the specific productivity of batch cultures. This revised version was published online in July 2006 with corrections to the Cover Date.     

Strategies for selecting recombinant CHO cell lines for cGMP manufacturing: Improving the efficiency of cell line generation

Transfectants with a wide range of cellular phenotypes are obtained during the process of cell line generation. For the successful manufacture of a therapeutic protein, a means is required to identify a cell line with desirable growth and productivity characteristics from this phenotypically wide‐ranging transfectant population. This identification process is on the critical path for first‐in‐human studies. We have stringently examined a typical selection strategy used to isolate cell lines suitable for cGMP manufacturing. One‐hundred and seventy‐five transfectants were evaluated as they progressed through the different assessment stages of the selection strategy. High producing cell lines, suitable for cGMP manufacturing, were identified. However, our analyses showed that the frequency of isolation of the highest producing cell lines was low and that ranking positions were not consistent between each assessment stage, suggesting that there is potential to improve upon the strategy. Attempts to increase the frequency of isolation of the 10 highest producing cell lines, by in silico analysis of alternative selection strategies, were unsuccessful. We identified alternative strategies with similar predictive capabilities to the typical selection strategy. One alternate strategy required fewer cell lines to be progressed at the assessment stages but the stochastic nature of the models means that cell line numbers are likely to change between programs. In summary, our studies illuminate the potential for improvement to this and future selection strategies, based around use of assessments that are more informative or that reduce variance, paving the way to improved efficiency of generation of manufacturing cell lines. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

A comparison of orbitally‐shaken and stirred‐tank bioreactors: pH modulation and bioreactor type affect CHO cell growth and protein glycosylation

Orbitally shaken bioreactors (OSRs) support the suspension cultivation of animal cells at volumetric scales up to 200 L and are a potential alternative to stirred‐tank bioreactors (STRs) due to their rapid and homogeneous mixing and high oxygen transfer rate. In this study, a Chinese hamster ovary cell line producing a recombinant antibody was cultivated in a 5 L OSR and a 3 L STR, both operated with or without pH control. Effects of bioreactor type and pH control on cell growth and metabolism and on recombinant protein production and glycosylation were determined. In pH‐controlled bioreactors, the glucose consumption and lactate production rates were higher relative to cultures grown in bioreactors without pH control. The cell density and viability were higher in the OSRs than in the STRs, either with or without pH control. Volumetric recombinant antibody yields were not affected by the process conditions, and a glycan analysis of the antibody by mass spectrometry did not reveal major process‐dependent differences in the galactosylation index. The results demonstrated that OSRs are suitable for recombinant protein production from suspension‐adapted animal cells. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1174–1180, 2016     

Process parameter shifting: Part I. Effect of DOT, pH, and temperature on the performance of Epo-Fc expressing CHO cells cultivated in controlled batch bioreactors

The impact of process environment changes on process performance is one of the most crucial process safety issues when cultivating mammalian cells in a bioreactor. In contrast, directed shifting of process parameters can also be used as an optimization tool providing higher cell and product yields. Compared to other strategies that also aim on the regulation of cell growth and protein expression process parameter shifts can be easily performed without reagent addition or even genetic modification of the host cell line. However, a successful application of changing process conditions implies a profound understanding of the provoked physiological changes within the cells. In a systematic approach we varied the dissolved oxygen tension (DOT), pH, and temperature of CHO cultures in controlled bioreactors and investigated the impact on growth, productivity, metabolism, product quality and cell cycle distribution using a recombinant CHO cell line expressing the highly glycosylated fusion protein Epo-Fc. We found the reduction of cultivation temperature and the reduction of (external) pH to exert the most significant effects on process performance by mainly reducing cell growth and metabolism. With respect to the cell line used we identified a set of parameters capable of affecting cell proliferation in favor of an increased specific productivity and total product yield. The well directed alteration of the process environment has emerged as a tool adequate for further process optimization applying a biphasic cultivation strategy.     

Comparison of the production of a human monoclonal antibody against HIV-1 by heterohybridoma cells and recombinant CHO cells: A flow cytometric study

The production of human monoclonal antibodies for therapeutic use is of increasing importance for treatment of viral infections such as AIDS. As human x mouse heterohybridomas rarely reach the growth rates and cell specific production rates of mouse hybridomas the transfection of standard cell lines, such as CHO or BHK, is a promising alternative. This has the additional advantage that the IgG subtype can be changed to suit the desired application. However, the use of a cell line that has not originally developed to produce antibodies, as lymphocytes and myeloma cells have, might have unrecognised drawbacks. This will be especially significant in the case of antibodies as each molecule consists of 4 chains linked by disulphide bonds which require specific intracellular factors to be properly folded and processed (Heavy chain binding protein, Protein Disulfide Isomerase a.o.). In this study we have therefore compared two cell lines: a human x mouse heterohybridoma producing IAM-2F5, a human IgG₃ antibody specific for HIV-1 with neutralising properties and a Chinese Hamster Ovary cell transfected with dihydrofolate reductase and with the heavy and light chain genes of IAM-2F5 modified to IgG₁. From each cell line three subclones were selected with low, medium and high specific production rates. Batch cultures were performed and the following cellular parameters analysed by flow cytometry; 1) total RNA content (translational activity); 2) total protein content; 3) cell cycle phase distribution; 4) concentration of light and heavy chains; 5) concentration of helper proteins such as BiP and PDI. The production rate of heterohybridoma cells was best reflected in the intracellular concentration of kappa chain, while the gamma chain concentration was comparable for all three subclones. In the CHO cells the gamma chain expression and thus gene copy number appeared to be the limiting factor. The GRP78/BiP concentration in CHO remained unchanged in spite of a 5-fold higher concentration of gamma chain in the high producing subclone. The PDI concentration in CHO cells was much lower compared to the heterohybridoma cells, irrespective of production rates.Abbreviations PDI protein disulfide isomerase - GRP78/BiP Glucose regulated protein; Heavy chain binding protein     

Development and manufacturability assessment of chemically‐defined medium for the production of protein therapeutics in CHO cells

Advantages of using internally developed chemically‐defined (CD) media for cell culture‐based therapeutic protein production over commercial media include better raw material control and medium vendor options, and most importantly, flexibility for process development and subsequent optimization needed for therapeutic protein production. Through several rounds of design of experiment (DOE) screening, and medium component supplementation and optimization studies, we successfully developed a CD basal medium (CDM) for CHO cell culture. The internally prepared liquid CDM demonstrated comparable cell culture performance to that from a commercially available control medium. However, when the same CDM formulation was transferred to two major commercial medium suppliers for manufacturing, cell culture performance utilizing these newly prepared media was significantly reduced compared with the in‐house prepared counterpart. An investigation was launched to assess whether key medium components were sensitive to large‐scale preparation of the final bulk media by the vendors. Further work necessitated the reformulation of the original CDM formulation into a core medium that was suitable for large‐scale media manufacturing. The modified preparation of the core medium with two separate supplements to generate the final CDM was able to recover the expected cell culture performance and monoclonal antibody (mAb) productivity. Confirmation of cell culture robustness in cell growth and production was corroborated in two additional mAb‐expressing cell lines. This work demonstrates that a robust CD medium is not only one that performs during the development stage, but also one that must be reproducible by commercial media vendors. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1163–1171, 2015     

Glycan Residues Balance Analysis - GReBA: A novel model for the N-linked glycosylation of IgG produced by CHO cells

The structure of N-linked glycosylation is a very important quality attribute for therapeutic monoclonal antibodies. Different carbon sources in cell culture media, such as mannose and galactose, have been reported to have different influences on the glycosylation patterns. Accurate prediction and control of the glycosylation profile are important for the process development of mammalian cell cultures. In this study, a mathematical model, that we named Glycan Residues Balance Analysis (GReBA), was developed based on the concept of Elementary Flux Mode (EFM), and used to predict the glycosylation profile for steady state cell cultures. Experiments were carried out in pseudo-perfusion cultivation of antibody producing Chinese Hamster Ovary (CHO) cells with various concentrations and combinations of glucose, mannose and galactose. Cultivation of CHO cells with mannose or the combinations of mannose and galactose resulted in decreased lactate and ammonium production, and more matured glycosylation patterns compared to the cultures with glucose. Furthermore, the growth rate and IgG productivity were similar in all the conditions. When the cells were cultured with galactose alone, lactate was fed as well to be used as complementary carbon source, leading to cell growth rate and IgG productivity comparable to feeding the other sugars. The data of the glycoprofiles were used for training the model, and then to simulate the glycosylation changes with varying the concentrations of mannose and galactose. In this study we showed that the GReBA model had a good predictive capacity of the N-linked glycosylation. The GReBA can be used as a guidance for development of glycoprotein cultivation processes.     

Specific inhibition of caspase-8 and -9 in CHO cells enhances cell viability in batch and fed-batch cultures

In an attempt to investigate the molecular mechanism that leads to apoptotic death in Chinese hamster ovary (CHO) cells in batch and fed-batch cultures, we cloned caspase-2, -8 and -9 from a CHO cDNA library. Recombinant Chinese hamster caspase-2 and -9 expressed in Escherichia coli show highest activities towards commercial peptide substrates Ac-VDVAD-pNA and Ac-LEHD-pNA, the designated commercial substrates for human caspase-2 and -9, respectively. However, Chinese hamster caspase-8 shows a broad specificity profile and it cleaves the caspase-9 substrate more efficiently than it cleaves the caspase-8 substrate. The commercially available fluoromethyl ketone type of caspase inhibitors, such as Z-LEHD-fmk, Z-IETD-fmk, Z-VDVAD-fmk and Z-DEVD-fmk, were shown to completely lack specificity in inhibiting these caspases. The reversible aldehyde form of inhibitors for human caspase-8 and -9, Ac-LEHD-CHO and Ac-IETD-CHO, are equally efficient in inhibiting Chinese hamster caspase-8. Therefore, the wildly used method of utilizing the "caspase-specific" inhibitors to track the role of individual caspases in dying cells can be inaccurate and thus misleading. As an alternative, we stably expressed dominant negative (DN) mutants of Chinese hamster caspase-2, -8 and -9 to specifically inhibit these enzymes in CHO cells. Our results showed that inhibition of either endogenous caspase-8 or caspase-9 enhanced the viability of the CHO cells in both batch and fed-batch suspension cultures, but the inhibition of caspase-2 had minimal effects. These results suggest that caspase-8 and -9 are possibly involved in the apoptotic cell death in batch and fed-batch cultures of CHO cells, whereas caspase-2 is not. These findings can be valuable in the development of strategies for genetically engineering CHO cells to counter apoptotic death in batch and fed-batch cultures.     

Effect of hydrocortisone on the production and glycosylation of an Fc-fusion protein in CHO cell cultures

Glucocorticoids are known to modulate various cellular functions such as cell proliferation, metabolism, glycosylation, and secretion of many proteins. We tested the effect of hydrocortisone (HC) on cell growth, viability, metabolism, protein production, and glycosylation of an Fc-protein expressing Chinese hamster ovary (CHO) cell culture. HC extended cell viability but impaired cell growth. The inhibitory effect on cell growth was dose-dependent and decreased when the glucocorticoid addition was delayed. When HC was added after 2 or 3 days of culture, an increase in glutamate consumption was observed, which was reversed by the glucocorticoid receptor antagonist mifepristone (Mif). Titer and specific productivity increased in the presence of HC. The increase in titer was only slightly reversed by Mif. On the other hand, Mif by itself induced an increase in titer to a level comparable to or higher than HC. Protein glycosylation was altered by the glucocorticoid in a dose- and time-dependent manner, with a shift to more acidic bands, which correlated with an increase in sialic acid moieties. This increase, which was not linked to a decrease in extracellular sialidase activity in HC-treated cultures, was reversed by Mif. Predictive models based on design of experiments enabled the definition of optimal conditions for process performance in terms of viability and titer and for the quality of the Fc-fusion protein in terms of glycosylation. The data obtained suggest a use of glucocorticoids for commercial production of Fc-fusion proteins expressed in CHO cells.     

Comparison of the endogenous IK currents in rat hippocampal neurons and cloned Kv2.1 channels in CHO cells

The Kv2.1 potassium channel is a principal component of the delayed rectifier I(K) current in the pyramidal neurons of cortex and hippocampus. We used whole-cell patch-clamp recording techniques to systemically compare the electrophysiological properties between the native neuronal I(K) current of cultured rat hippocampal neurons and the cloned Kv2.1 channel currents in the CHO cells. The slope factors for the activation curves of both currents obtained at different prepulse holding potentials and holding times were similar, suggesting similar voltage-dependent gating. However, the half-maximal activation voltage for I(K) was approximately 20 mV more negative than the Kv2.1 channel in CHO cells at a given prepulse condition, indicating that the neuronal I(K) current had a lower threshold for activation than that of the Kv2.1 channel. In addition, the neuronal I(K) showed a stronger holding membrane potential and holding time-dependence than Kv2.1. The Kv2.1 channel gave a U-shaped inactivation, while the I(K) current did not. The I(K) current also had much stronger voltage-dependent inactivation than Kv2.1. These results imply that the neuronal factors could make Kv2.1 channels easier to activate. The information obtained from these comparative studies help elucidate the mechanism of molecular regulation of the native neuronal I(K) current in neurons.     

Differing effects of isoleucine deficiency on the toxicity of MNNG for 10T1/2 and CHO cells

Summary Isoleucine deficiency sensitizes C3H 10T1/2 cells to the cytotoxic effects of MNNG. Synchrony of proliferation is not required for this effect since it occurs prior to full growth arrest and subsequent establishment of synchronous proliferation after refeeding in complete medium. Furthermore, confluence arrest of proliferation of 10T1/2 cells does not affect their cytotoxic response to MNNG, although they proliferate synchronously after replating at low density. In contrast, the toxicity of MNNG for CHO cells is not altered by isoleucine deficiency, even though these cells are readily synchronized by refeeding in complete medium after transient isoleucine deficiency. This research was supported by Grant BC-142 from the American Cancer Society and Grants CA-16086 and CA-17973 from the National Cancer Institute.     

Development of serum-free medium supplemented with hydrolysates for the production of therapeutic antibodies in CHO cell cultures using design of experiments

An efficient method of formulating serum-free medium (SFM) for production of therapeutic antibodies by recombinant CHO (rCHO) cells was developed using two rCHO cell lines producing a therapeutic antibody. In this method, ten kinds of SFM were prepared by supplementing the basal SFM with statistically designed mixtures (total 5 g L^?1) of three non-animal-derived hydrolysates: yeastolate, soy hydrolysate, and wheat gluten hydrolysate. When the two rCHO cell lines were cultivated, the mixtures of soy hydrolysate and wheat gluten hydrolysate showed a positive effect on cell growth. On the other hand, the mixtures including a high portion of yeastolate significantly enhanced specific antibody productivity. To reconstitute the mixture ratios of the three hydrolysates for high growth and antibody production, the effect of each medium was analyzed by the statistical program Design-Expert®. The resulting medium gave a 1.9–3.3-fold increase in the maximum antibody concentration, compared to the basal SFM. Taken together, the supplementation of hydrolysates to the basal SFM with the help of statistical analysis is an efficient means of developing SFM for therapeutic antibody production by rCHO cells.     

Effect of cloned gene dosage on cell growth and hepatitis B surface antigen synthesis and secretion in recombinant CHO cells

The effect of cloned gene copy number on growth and product formation has been studied in sufficient detail using a Chinese hamster overy (CHO) cell line producing recombinant hepatits B surface antigen (HbsAg). Batch culture experiments were carried out in T flasks in order to characterize cell growth and HbsAg secretion in various clones carrying different numbers of HbsAg gene copies integrated into CHO cell chromosomes. Specific growth rates were found to decrease with increasing gane copy number. Secreted HbsAg concentration and specific HbsAg secretion rates were found to increase with increase in gene copy number. Gene copy numbers in each clone determined using Southern hybridizations were positively correlated with intracellular dihydrofolate reductase (dhfr) content using a flow cytometric assay. The mRNA levels quantitated using Northern hybridization followed by autroadiography and densitometry also gave the same trends. The flow cytometry experiments show that while parental cells were quite homogeneous with respect to intracellular dhfr content, the amplified clones exhibit a great deal of heterogeneity in dhfr content. Pulse-chase experiments show that the efficiency of HbsAg secretion (defined here as the fraction of initially labeled HbsAg that is secreted into the extracellular medium at the end of a 23.5-h chase) decreases and also that the intracellular HbsAg degradation increases with increasing gene copy number.     

Anti‐cell death engineering of CHO cells: Co‐overexpression of Bcl‐2 for apoptosis inhibition,Beclin‐1 for autophagy induction

Genetic engineering approaches to inhibit cell death in Chinese hamster ovary (CHO) cell cultures have been limited primarily to anti‐apoptosis engineering. Recently, autophagy has received attention as a new anti‐cell death engineering target in addition to apoptosis. In order to achieve a more efficient protection of cells from the stressful culture conditions, the simultaneous targeting of anti‐apoptosis and pro‐autophagy in CHO cells (DG44) was attempted by co‐overexpressing an anti‐apoptotic protein, Bcl‐2, and a key regulator of autophagy pathway, Beclin‐1, respectively. Co‐overexpression of Bcl‐2 and Beclin‐1 exhibited a longer culture period as well as higher viability during serum‐free suspension culture, compared with the control (without co‐overexpression of Bcl‐2 and Beclin‐1) and Bcl‐2 overexpression only. In addition to the efficient inhibition of apoptosis by Bcl‐2 overexpression, Beclin‐1 overexpression successfully induced the increase in the autophagic marker protein, LC3‐II, and autophagosome formation with the decrease in mTOR activity. Co‐immunoprecipitation and qRT‐PCR experiments revealed that the enforced expression of Beclin‐1 increased Ulk1 expression and level of free‐Beclin‐1 that did not bind to the Bcl‐2 despite the Bcl‐2 overexpression. Under other stressful culture conditions such as treatment with sodium butyrate and hyperosmolality, co‐overexpression of Bcl‐2 and Beclin‐1 also protected the cells from cell death more efficiently than Bcl‐2 overexpression only, implying the potential of autophagy induction. Taken together, the data obtained here provide the evidence that pro‐autophagy engineering together with anti‐apoptosis engineering yields a synergistic effect and successfully enhances the anti‐cell death engineering of CHO cells. Biotechnol. Bioeng. 2013; 110: 2195–2207. © 2013 Wiley Periodicals, Inc.     

The effect of the dilution rate on CHO cell physiology and recombinant interferon-gamma production in glucose-limited chemostat culture

The physiology of a recombinant Chinese hamster ovary cell line in glucose-limited chemostat culture was studied over a range of dilution rates (D = 0.008 to 0.20 h(-1)). The specific growth rate (mu) deviated from D at low dilution rates due to an increased specific death rate. Extrapolation of these data suggested a minimum specific growth rate of 0.011 h(-1) (mu(max) = 0.025 h(-1)) The metabolism at each steady state was characterized by determining the metabolic quotients for glucose, lactate, ammonia, amino acids, and interferon-gamma (IFN-gamma). The specific rate of glucose uptake increased linearly with mu, and the saturation constant for glucose (K(s)) was calculated to be 59.6 muM. There was a linear increase in the rate of lactate production with a higher yield of lactate from glucose at high growth rates. The decline in the rate of production of lactate, alanine, and serine at low growth rate was consistent with the limitation of the glycolytic pathway by glucose. The specific rate of IFN-gamma production increased with mu in a manner indicative of a growth-related product. Despite changes in the IFN-gamma production rate and cell physiology, the pattern of IFN-gamma glycosylation was similar at all except the lowest growth rates where there was increased production of nonglycosylated IFN-gamma. (c) 1993 John Wiley & Sons, Inc.     

Exometabolome profiling reveals activation of the carnitine buffering pathway in fed-batch cultures of CHO cells co-fed with glucose and lactic acid

Adjustments to CHO cell physiology were recently observed during implementation of a Raman spectroscopy-based glucose and lactate control strategy. To further understand how these cells, under monoclonal antibody (mAb) production conditions, utilized the extra lactic acid fed, we performed a comprehensive semi-quantitative and time-dependent analysis of the exometabolome. This study focused on the CHO cell's metabolic shift from the fifth day of culture. We compared relative levels of extracellular metabolites in the absence or presence of a 2 g/L lactic acid setpoint while glucose was kept at 4 g/L. Our hypothesis is that extra lactic acid would supply more pyruvate, favoring oxidative phosphorylation. We subsequentially uncovered several carnitine derivatives as biomarkers of the simultaneous activation of TCA anaplerotic pathways as well as a carbon-buffering pathway. CHO cells exhibited a balance between intermediates from (i) amino acid catabolism, (ii) fatty acid β-oxidation, and (iii) pyruvate from glycolysis and lactic acid; and the secretion of their intermediate carnitine derivatives. In addition, 3-hydroxy-methyl-glutaric acid (HMG) and mevalonate syntheses were found as biomarkers of alternative acyl group removal. Together, under a limited capacity to assimilate the surplus of acyl-CoA groups as well as an ability to maintain the acyl-CoA: free CoA ratio for proper and continuous functioning of the TCA cycle, CHO cells activate the carnitine-buffering system, HMG, and mevalonate pathways.     

Overexpression of the mitochondrial pyruvate carrier reduces lactate production and increases recombinant protein productivity in CHO cells

Chinese hamster ovary (CHO) cells are characterized by a low glucose catabolic efficiency, resulting in undesirable lactate production. Here, it is hypothesized that such low efficiency is determined by the transport of pyruvate into the mitochondria. The mitochondrial pyruvate carrier (MPC), responsible for introducing pyruvate into the mitochondria, is formed by two subunits, MPC1 and MPC2. Stable CHO cell lines, overexpressing the genes of both subunits, were constructed to facilitate the entry of pyruvate into the mitochondria and its incorporation into oxidative pathways. Significant overexpression of both genes, compared to the basal level of the control cells, was verified, and subcellular localization of both subunits in the mitochondria was confirmed. Kinetic evaluation of the best MPC overexpressing CHO cells showed a reduction of up to 50% in the overall yield of lactate production with respect to the control. An increase in specific growth rate and maximum viable cell concentration, as well as an increase of up to 40% on the maximum concentration of two recombinant model proteins transiently expressed (alkaline phosphatase or a monoclonal antibody), was also observed. Hybrid cybernetic modeling, that considered 89 reactions, 25 extracellular metabolites, and a network of 62 intracellular metabolites, explained that the best MPC overexpression case resulted in an increased metabolic flux across the mitochondrial membrane, activated a more balanced growth, and reduced the Warburg effect without compromising glucose consumption rate and maximum cell concentration. Overall, this study showed that transport of pyruvate into the mitochondria limits the efficiency of glucose oxidation, which can be overcome by a cell engineering approach.     

Methyl mercury uptake and associations with the induction of chromosomal aberrations in Chinese hamster ovary (CHO) cells

In order to evaluate possible health effects of environmental exposure of humans towards methyl mercury species, relevant exposure experiments using methyl mercury chloride in aqueous solution and Chinese hamster ovary (CHO) cells were performed. The solution was monitored for the presence of monomethyl, dimethyl and elemental mercury by several analytical techniques including chromatographic as well as atomic absorption and mass spectrometric methods. Methyl mercury induces structural chromosomal aberrations (CA) and sister chromatid exchanges (SCE) in CHO cells. At a concentration of methyl mercury in the culture medium of 1.0 x 10(-6) M where the frequencies of CA and SCE are significantly elevated, the intracellular concentration was 1.99 x 10(-16) mol/cell. Possible biochemical processes leading to the cytogenetic effects are discussed together with toxicological consequences, when humans (e.g. workers at waste deposits) are exposed to environmental concentrations of methyl mercury.